Interferon is potentially a very valuable therapeutic agent in the treatment of neoplastic or viral disease states. The development of this substance into a widely available drug has been hampered by the difficulty of producing and isolating this active compound in reasonable amounts. An important aid in developing procedures for the large scale production, purification and ultimate characterization of human interferon would be the availability of an assay which could be completed in less than 24 hours.
Similarly, the presently available interferon assays are far too slow to allow practical clinical applications. It is known that blood levels of interferon are elevated above normal levels in the presence of a viral infection. Assaying of a subject's interferon levels allows the physician to determine whether a viral agent is the causative agent and allows the physician to make a rational choice in the selection of appropriate therapy. Information gained from such an assay could provide the basis for avoiding unnecessary, expensive and potentially harmful antibiotic therapy on a patient who is suffering from a viral disease which would normally be unresponsive to such therapy. Again, a critical aspect of such an assay would be the rapidity with which useful information could be made available to the physician from the time the sample is drawn. A period of three days would obviously be much too long, since it is unlikely that therapy could or should be withheld from a patient for such extended period.
A number of procedures have been known in the art to assay for interferon levels. Such assays are based on the inhibition of virus growth and measurable aspects thereof such as, for example, the virus yield, plaque number, cytopathic effect or viral products. There is presently no standard assay procedure. In general, the titers (50% end points) derived from the various assay methods are related to an internationally accepted reference preparation. The currently utilized reference is a human leukocyte interferon having a concentration of 5,000 units/ml which is held by the Medical Research Council, London, England and by the National Institutes of Health, Bethesda, Maryland.
Examples of some specific disclosed assays for interferon include a publication by Lindenmann and Gifford, Virology 19, 302 (1963) which discloses a plaque inhibition assay and a paper by John A. Armstrong, Applied Microbiology 21, No. 4, 723 (1971) which described a semi-micro dye binding assay for rabbit interferon employing quantitation of inhibition of cytopathic effects. In the Lindenmann and Gifford assay, both interferon and challenge virus were added simultaneously to chick embryo cell monolayers in bottles with an agar overlay and without the need of an absorption period of either interferon or virus. Since interferon diffuses faster than virus, and since virus was adsorbed to less than 1% of the initial target cells, all cells having been exposed to interferon under circumstances of the assay, the cells were effectively treated with interferon prior to virus exposure. After 44 to 48 hours of incubation at 37.degree. C., the monolayers were stained with crystal violet and the plaques counted.
In the Armstrong procedure, confluent monolayer cultures of weanling rabbit kidney cells were exposed to interferon dilutions, challenged with virus, and the culture cells were stained with methylrosaniline chloride. The bound dye was then eluted and measured colorimetrically. It should be noted that the test samples, cell culture and interferon standards were incubated overnight, and after removal of the incubation medium which contained interferon, the cultured cells were treated with the challenge virus (vesicular stomatitis virus) and then incubated an additional 24 hours, or even longer, until the virus controls showed 50-90% cytopathic effects microscopically. Additional steps were required to introduce and fix the dye and to dry the preparation. It is evident that it is not possible to read the results of the assay before the third day after the procedure is started and possibly even later.
In each of the foregoing assay procedures, the test sample was added to monolayers. It has heretofore been generally believed, in the prior art, that confluent monolayers were of critical necessity to provide cells of sufficient uniformity to avoid variations in results which might be attributable to variations in cell density and/or quality of cells. It was also felt that variations in incubation times would change the susceptibility of the cells to interferon or the challenge virus in ways that would adversely affect the reproductibility and uniformity of assay results (Interferon and its Clinical Potential, D. A. J. Tyrell, William Heinemen Medical Books Ltd., London, 1976). In accordance with the present invention, it has unexpectedly been found that the total time for the bioassay procedure can be substantially reduced by introducing the cell culture in the form of a suspension, as opposed to the confluent monolayers thought to be essential in the prior art. In this manner, it has been found possible to add the challenge virus and the cells to the serially diluted interferon sample without the need of any prior incubation of the interferon samples and the cells.